The aim of this new project is twofold: 1) to characterize differences between tumor and normal breast microvasculature in a rat model and in human cancer, 2) to study interactions between tumor cells and microvascular endothelial cells (EC) derived from the primary tumor as well as from the common sites of metastasis, such as lungs, adrenals, and bone marrow. Attention has also been focused on elucidating the culture conditions for myoepithelial and luminal epithelial cells from rat mammary tissue. Lactation fluid (LF) is a rich and uncontaminated source of terminal ductal epithelial cells and as such has produced cells tentatively identified as epithelial cells. The studies to date have been successful in determining an effective tissue disaggregation technique, culture system, and selective growth media for the different cell types. The utilization of flow cytometry to isolate these cell populations will greatly increase the purity of the cell populations obtained. When combined with the knowledge gained from the present studies, it may allow the routine culture of the cell types of interest. We have successfully grown rat mammary pad microvascular EC (20% pure). Cell cultures and clones were obtained from LF, adrenal, and lung tissues and are currently being further characterized. To establish cell lines with longer in vitro life span, immortalization of the cell populations was routinely performed using SV40 large T antigen and HPV 16 E6 and E7 genes. A number of cell lines have been stably transfected with these constructs and their characteristics are being investigated. The cell types listed here will provide the building blocks for studying tumor-epithelial, EC interactions in the initiation, progression, and metastasis of breast carcinoma.